The present invention relates to a crystal of a cation channel protein, and methods of using such a crystal in screening potential drugs and therapeutic agents for use in treating conditions related to the function of such channels in vivo.
Although numerous types of channel proteins are known, the main types of ion channel proteins are characterized by the method employed to open or close the channel protein to either permit or prevent specific ions from permeating the channel protein and crossing a lipid bilayer cellular membrane. One important type of channel protein is the voltage-gated channel protein, which is opened or closed (gated) in response to changes in electrical potential across the cell membrane. Another type of ion channel protein are celled mechanically gated channel proteins, for which a mechanical stress on the protein opens or closes the channel. Still another type is called a ligand-gated channel, which opens or closes depending on whether a particular ligand is bound the protein. The ligand can be either an extracellular moiety, such as a neurotransmitter, or an intracellular moiety, such as an ion or nucleotide.
Presently, over 100 types of ion channel proteins have been described, with additional ones being discovered. Basically, all ion channels have the same basic structure regarding the permeation of their specific ion, although different gating mechanisms (as described above) can be used. One of the most common types of channel proteins, found in the membrane of almost all animal cells, permits the specific permeation of potassium ions (K+) across a cell membrane. In particular, potassium ions permeate rapidly across cell membranes through K+ channel proteins (up to 108 ions per second). Moreover, potassium channel proteins have the ability to distinguish among potassium ions, and other small alkali metal ions, such as Li+ or Na+ with great fidelity. In particular, potassium ions are at least ten thousand times more permanent than sodium ions. In light of the fact that both potassium and sodium ions are generally spherical in shape, with radii of about 1.33 xc3x85 and 0.95 xc3x85 respectively, such selectivity is remarkable.
Broadly, potassium channel proteins comprise four (usually identical) subunits. Presently two major types of subunits are known. One type of subunit contains six long hydrophobic segments (presumably membrane-spanning), while the other type contains two hydrophobic segments. Regardless of what type of subunits are used, potassium channel proteins are highly selective for potassium ions, as explained above.
Among their many functions, potassium channel proteins control the pace of the heart, regulate the secretion of hormones such as insulin into the blood stream, generate electrical impulses underlying information transfer in the nervous system, and control airway and vascular smooth muscle tone. Thus, potassium channels participate in cellular control processes that are abnormal, such as cardiac arrhythmia, diabetes mellitus, seizure disorder, asthma and hypertension, to name only a few.
Although potassium channel proteins are involved in such a wide variety of homeostatic functions, few drugs or therapeutic agents are available that act on potassium channel proteins to treat abnormal processes. A reason for a lack of presently available drugs that act on potassium channel proteins is that isolated potassium channel proteins are not available in great abundance, mainly because an animal cell requires only a very limited number of such channel proteins in order to function. Consequently, it has been very difficult to isolate and purify potassium channel proteins, reducing the amount of drug screening efforts in search of potassium channel protein acting drugs.
Hence, what is needed is accurate information regarding the structure of cation channel proteins so that drugs or therapeutic agents having an appropriate structure to potentially interact with a cation channel protein can be selected.
What is also needed is an ability to overcome the physical limitations regarding the isolation and purification of cation channel proteins, particularly potassium ion channel proteins.
What is also needed is a reliable method of utilizing cation channel proteins in screening potential drugs or agents for their possible use in treating conditions related to the function of cation channel proteins in vivo.
What is also needed are novel methods of using accurate information regarding the structure of cation channel proteins so that drugs or therapeutic agents can be screened for potential activity in treating abnormal control processes of the body.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
There is provided, in accordance with the present invention, a method of preparing a functional cation channel protein for use in an assay for screening potential drugs or other agents which interact with a cation channel protein, which permits the screening of potential drugs or agents that may be used as potential therapeutic agents in treating conditions related to the function of cation channel proteins in vivo.
More specifically, the method comprising the steps of providing a functional cation channel protein, conjugating the functional cation channel protein to a solid phase resin, contacting the potential drug or agent to the functional cation channel protein conjugated to the solid phase resin, removing the functional cation channel protein from the solid phase resin, and determining whether the potential drug or agent is bound to the cation channel protein.
In particular, the present invention extends to a method of preparing a functional cation channel protein for use in an assay as described above, wherein the providing step of the method comprises expressing an isolated nucleic acid molecule encoding the cation channel protein in a unicellular host, such that the cation channel protein is present in the cell membrane of the unicellular host, lysing the unicellular host in a solubilizing solution so that the cation channel protein is solubilized in the solution, and extracting the cation channel protein from the solubilizing solution with a detergent. In a preferred embodiment, the isolated nucleic acid molecule comprises a DNA sequence of SEQ ID NO:17, or degenerate variants thereof, or an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated nucleic acid molecule having a DNA sequence of SEQ ID NO:17, or degenerate variants thereof.
Numerous methods of lysing a unicellular host are known to the skilled artisan, and have applications in the present invention. In a preferred embodiment, lysing the unicellular host in a solubilizing solution comprises sonicating the unicellular host in a protein solubilizing solution comprising 50 mM Tris buffer, 100 mM KCl, 10 mM MgSO4, 25 mg DNAse, 1, 250 mM sucrose, pepstatin, leupeptin, and PMSF, pH 7.5.
Furthermore, a skilled artisan is aware of numerous detergents that can be used to extract an integral membrane bound protein, such as a cation channel protein, from a solubilizing solution described above. Examples of such detergents include SDS, Triton-100, Tween 20, Tween 80, glycerol, or decylmaltoside, to name only a few. Preferably, 40 mM decylmaltoside is used to extract the cation channel protein from the solubilizing solution.
Moreover, numerous solid phase resins to which a functional cation channel protein can be conjugated have applications in a method of preparing a functional cation channel protein for use in an assay, as described above. For example, a solid phase resin comprising insoluble polystyrene beads, PVF, polyethylene glycol, or a cobalt resin, to namely only a few have application in the present invention. Preferably, a cation channel protein is conjugated to a cobalt resin at a protein to resin ratio that allows for saturation of the resin with the cation channel protein. Moreover, after conjugation, the cobalt resin is preferably used to line a column having a volume of about 1 ml.
After the cation channel protein is conjugated to a solid phase resin, it is contacted with a potential drug or agent, which is given an opportunity to bind to cation channel protein.
Subsequently, the cation channel protein is removed from the solid phase resin, and analyzed to determine whether the potential drug or agent is bound thereto. Numerous methods of removing the cation channel protein from the solid phase resin are known to those of ordinary skill in the art. In a preferred embodiment, wherein the solid phase resin is a cobalt resin, the removing step comprises contacting the cation channel protein conjugated to the solid phase resin with an imidazole solution. This solution readily cleaves any bonds conjugating the cation channel protein to the resin, so that the protein can removed from the resin, and collected for further analysis to determine whether the potential drug or agent is bound to the protein.
After the cation channel protein has been removed from the resin, it must be examined to determine whether the potential drug or agent is bound thereto. If bound, the drug or agent may have uses involved in modulation of the function of a cation channel protein in vivo, including uses as a therapeutic agent in treating conditions related to the function of cation channel proteins. Numerous analytical methods are presently available to the skilled artisan for determining whether the potential ligand is bound to the cation channel protein. Examples of such methods include molecular weight analysis with SDS-PAGE, immunoassays using an antibody to the drug or agent, HPLC, or mass spectrometry.
Furthermore, the present invention extends to a method of using a functional cation channel protein in an assay for screening potential drugs or agents which interact with the cation channel protein, wherein the potential drug or agent is a member of a library of compounds, which is contacted to the cation channel protein. Examples of libraries having applications in the present invention include, but are not limited to, a mixture of compounds, or a combinatorial library of compounds. Furthermore, examples of combinatorial compounds having applications in the present invention include, but are not limited to, a phage display library, or a synthetic peptide library, to name only a few.
In another embodiment, the present invention extends to a prokaryotic cation channel protein mutated to mimic a functional eukaryotic cation channel protein. More specifically, Applicant has discovered that all cation channel proteins from all organisms have a conserved structure. Hence, placing mutations in a potassium channel from a prokaryotic organism, for example, can permit the use of the prokaryotic cation channel protein in screening assays for drugs that may interact with specific eukaryotic cation channel proteins. For example, a prokaryotic potassium channel protein can be mutated to mimic a cardiac potassium channel protein, a venous potassium channel protein, or a neuro potassium channel of a human, to name only a few.
Hence, pursuant to the present invention, a prokaryotic potassium channel protein, a prokaroytic sodium channel protein, or a prokaryotic calcium channel protein can be mutated to mimic a eukaryotic cation channel protein.
Examples of prokaryotic organisms from which a prokaryotic cation channel protein can be taken and mutated to mimic a eukaryotic cation channel protein include E. coli, Streptomyces lividans, Clostridium acetrobutylicum, or Staphylcoccus aureus, to name only a few. Furthermore, such prokaryotic cation channel proteins can comprise an amino acid sequence of SEQ ID Nos: 1, 2, 3, or 7, or conserved variants thereof. In a preferred embodiment, the prokaryotic cation channel protein mutated to mimic a eukaryotic cation channel protein, wherein the prokaryotic cation channel protein in a potassium channel protein from Streptomyces lividans. 
Furthermore, pursuant to the present invention, a prokaryotic cation channel protein can be mutated to mimic eukaryotic potassium channel protein, a eukaryotic sodium channel protein, or a eukaryotic calcium channel protein. Preferably, the eukaryotic cation channel protein is produced endogenously in a eukaryotic organism, such as an insect or a mammal, for example. More specifically, pursuant to the present invention, a prokaryotic cation channel protein is mutated to mimic a eukaryotic cation channel protein endogenously produced in a eukaryotic organism selected from the group consisting of Drosophila melanogaster, Homo sapiens, C. elegans, Mus musculus, Arabidopsis thaliana, paramecium tetraaurelia or Rattus novegicus, or having an amino acid sequence comprising SEQ ID Nos: 4, 5, 6, 8, 9, 10, 11, 12, 13, or 14, or conserved variants thereof.
In a preferred embodiment, the present invention extends to a prokaryotic cation channel protein mutated to mimic a functional eukaryotic channel protein, wherein the prokaryotic cation channel protein is a potassium channel protein from Streptomyces lividans comprising an amino acid sequence of SEQ ID NO:1 or degenerate variants thereof, and the eukaryotic cation channel is a potassium channel protein comprising an amino acid sequence of SEQ ID NO:4 or conserved variants thereof. As a result, the mutated prokaryotic channel protein comprises an amino acid sequence of SEQ ID NO:16, or conserved variants thereof, which is encoded by an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:17, or degenerate variants thereof.
In another embodiment, the present invention extends to a method of using a crystal of a cation channel protein, as described herein, in an assay system for screening drugs and other agents for their ability to modulate the function of a cation channel protein, comprising the steps of initially selecting a potential drug or agent by performing rational drug design with the three-dimensional structure determined for a crystal of the present invention, wherein the selecting is performed in conjunction with computer modeling. After potential drugs or agents have been selected, a cation channel protein is contacted with the potential drug or agent. If the drug or therapeutic agent has potential use for modulating the function of a cation channel protein, a change in the function of the cation channel after contact with the agent, relative to the function of a similar cation channel protein not contacted with the agent, or the function of the same cation channel protein prior to contact with the agent. Hence, the change in function is indicative of the ability of the drug or agent to modulate the function of a cation channel protein.
Furthermore, the present invention extends to extends to a method of using a crystal of a cation channel protein as described herein, in an assay system for screening drugs and other agents for their ability to modulate the function of a cation channel protein, wherein the crystal comprises a Naxe2x88x92 channel protein, a K+ channel protein, or a Ca2+ channel protein.
The present invention further extends to a method of using a crystal of a cation channel protein in an assay for screening drugs other drugs for their ability to modulate the function of a cation channel protein, wherein the crystal of the cation channel protein comprises an amino acid sequence of:
residues 23 to 119 of SEQ ID NO:1 (Streptomyces lividans);
residues 61 to 119 of SEQ ID NO:2 (E. coli);
residues 61 to 119 of SEQ ID NO:3 (Clostridium acetrobutylicum);
residues 61 to 119 of SEQ ID NO:4 (Drosophila melanogaster);
residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:7 (Paramecium tetraaurelia);
residues 61 to 119 of SEQ ID NO:8 (C. elegans);
residues 61 to 119 of SEQ ID NO:9 (Mus musculus);
residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:11 (Arabidopsis thaliana);
residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or
residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);
or conserved variants thereof.
In a preferred embodiment of a method of using a crystal of a cation channel protein in an assay for screening drugs or other agents for their ability to modulate the function of a cation channel protein, the crystal comprises a potassium channel protein, comprising amino acid residues 23 to 119 of SEQ ID NO:1, a space grouping of C2, and a unit cell of dimensions of a=128.8 xc3x85, b=68.9 xc3x85, c=112.0 xc3x85, and xcex2=124.6xc2x0.
Moreover, it is important to note that a drug""s or agent""s ability to modulate the function of a cation channel protein includes, but is not limited to, increasing or decreasing the cation channel protein""s permeability to the specific cation relative to the permeability of the same or a similar not contacted with the drug or agent, or the same cation channel protein prior to contact with the drug or agent.
In a further embodiment, the present invention extends to a method of using a crystal of a cation channel protein, as set forth herein, in an assay system for screening drugs and other agents for their ability to treat conditions related to the function of cation channel proteins in vivo, and particularly in abnormal cellular control processes related to the functioning of cation channel protein. Such a method comprises the initial step of selecting a potential drug or other agent by performing rational drug design with the three-dimensional structure determined for a crystal of the invention, wherein the selecting is performed in conjunction with computer modeling. After potential drugs or therapeutic agents are selected, a cation channel protein is contacted with the potential drug or agent. If an interaction of the potential drug or other agent with the cation channel is detected, it is indicative of the potential use of the drug or agent to treat conditions related the function of cation channel proteins in vivo. Examples of such conditions include, but are not limited to, cardiac arrhythmia, diabetes mellitus, seizure disorder, asthma or hypertension, to name only a few.
Furthermore, a crystal of a cation channel protein used in the method for screening drugs or agents for their ability to interact with a cation channel comprises an Na+ channel protein, K+ channel protein, or Ca2+ channel protein. Hence, the method of the present invention can be used to screen drugs or agents capable of treating conditions related to the function of such channels.
Moreover, the present invention extends to a crystal used in the method for screening drugs or agents for their ability to interact with a cation channel protein comprising an amino acid sequence of:
residues 23 to 119 of SEQ ID NO:1 (Streptomyces lividans);
residues 61 to 119 of SEQ ID NO:2 (E. coli);
residues 61 to 119 of SEQ ID NO:3 (Clostridium acetrobutylicum);
residues 61 to 119 of SEQ ID NO:4 (Drosophila melanogaster);
residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:7 (Paramecium tetraaurelia);
residues 61 to 119 of SEQ ID NO:8 (C. elegans);
residues 61 to 119 of SEQ ID NO:9 (Mus musculus);
residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:11 (Arabidopsis thaliana);
residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or
residues 61 to 119 of SEQ ID NO:14 (Homo sapiens),
or conserved variants thereof.
In a preferred embodiment, a crystal used in a method for screening drugs or agents for their ability to interact with a cation channel, comprises amino acid residues 23 to 119 of SEQ ID NO:1, has a space grouping of C2, and a unit cell of dimensions of a=128.8 xc3x85, b=68.9 xc3x85, c=112.0 xc3x85, and xcex2=124.6xc2x0.
In yet another embodiment, the present invention extends to a method of using a crystal of a cation channel protein described herein, in an assay system for screening drugs and other agents for their ability to permeate through a cation channel protein, comprising an initial step of selecting a potential drug or other agent by performing rational drug design with the three-dimensional structure determined for the crystal, wherein the selecting of the potential drug or agent is performed in conjunction with computer modeling. After a potential drug or agent has been selected, a cation channel protein can be prepared for use in the assay. For example, preparing the cation channel protein can include isolating the cation channel protein from the membrane of a cell, and then inserting the cation channel protein into a membrane having a first and second side which is impermeable to the potential drug or agent. As a result, the cation channel protein traverses the membrane, such that the extracellular portion of the cation channel protein is located on the firs side of the membrane, and the intracellular portion of the cation channel protein is located on the second side of the membrane. The extracellular portion of the cation channel membrane can then be contacted with the potential drug or agent. The presence of the drug or agent in the second side of the membrane is indicative of the drug""s or agent""s potential to permeate the cation channel protein, and the drug or agent is selected based on its ability to permeate the cation channel protein.
In addition, a crystal used in a method for screening drugs or agents for their ability to permeate a cation channel can comprise a Na+ channel protein, a K+ protein channel, or a Ca2+ protein channel.
Furthermore, the present invention extends to the use of a crystal in an assay system for screening drugs and other agents for their ability to permeate through a cation channel protein, wherein the crystal comprises an amino acid sequence of:
residues 23 to 119 of SEQ ID NO:1 (Streptomyces lividans);
residues 61 to 119 of SEQ ID NO:2 (E. coli);
residues 61 to 119 of SEQ ID NO:3 (Clostridium acetrobutylicum);
residues 61 to 119 of SEQ ID NO:4 (Drosophila melanogaster);
residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:7 (Paramecium tetraaurelia);
residues 61 to 119 of SEQ ID NO:8 (C. elegans);
residues 61 to 119 of SEQ ID NO:9 (Mus musculus);
residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:11 (Arabidopsis thaliana);
residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or
residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);
or conserved variants thereof.
In a preferred embodiment, the crystal used in an assay system of the present invention for screening drugs and other agents for their ability to permeate through a cation channel protein comprises amino acid residues 23 to 119 of SEQ ID NO:1, has a space grouping of C2, and a unit cell of dimensions of a=128.8 xc3x85, b=68.9 xc3x85, c=112.0 xc3x85, and xcex2=124.6xc2x0.
Naturally, the present invention extends to an isolated nucleic acid molecule encoding a mutant K+ channel protein, comprising a DNA sequence of SEQ ID NO:17, or degenerate variants thereof.
Furthermore, the present invention extends to an isolated nucleic acid molecule hybridizable to an isolated nucleic acid molecule encoding a mutant K+ channel protein under standard hybridization conditions.
Moreover, isolated nucleic acid molecules of the present invention, and described above, can be detectably labeled. Examples of detectable labels having applications in the present invention include, but are not limited to, radioactive isotopes, compounds which fluoresce, or enzymes.
The present invention further extends to an isolated nucleic acid molecule encoding a mutant K+ channel protein, or degenerate variants thereof, comprising an amino acid sequence of SEQ ID NO:16, or conserved variants thereof.
In addition, the present invention extends to an isolated nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:16, or conserved variants thereof, wherein the isolated nucleic acid molecule is hybridizable under standard hybridization conditions to an isolated nucleic acid molecule encoding a K+ channel protein, or degenerate variants thereof.
Furthermore, the present invention extends to a mutant cation channel protein comprising an amino acid sequence of SEQ ID NO:16, or conserved variants thereof.
In addition, the present invention extends to a cloning vector comprising an isolated nucleic acid molecule, or degenerate variants thereof, which encodes a mutant cation channel protein of the present invention, or conserved variants thereof, and an origin of replication. The present invention also extends to a cloning vector comprising an origin of replication and an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated nucleic acid molecule, or degenerate variants thereof, which encodes a mutant cation channel protein of the present invention.
Examples of cloning vectors having applications in the present invention include, but are not limited to, E. coli, bacteriophages, plasmids, and pUC plasmid derivatives. More specifically, examples of bacteriophages, plasmids, and pUC plasmid derivatives having applications herein comprise lambda derivatives, pBR322 derivatives, and pGEX vectors, or pmal-c, pFLAG, respectively.
Naturally, the present invention extends to an expression vector comprising an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:17, or degenerate variants thereof, operatively associated with a promoter. In another embodiment, an expression vector comprises an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated nucleic acid comprising a DNA sequence of SEQ ID NO:17, or degenerate variants thereof, operatively associated with a promoter.
Examples of promoters having applications in expression vectors of the present invention comprise immediate early promoters of hCMV, early promoters of SV40, early promoters of adenovirus, early promoters of vaccinia, early promoters of polyoma, late promoters of SV40, late promoters of adenovirus, late promoters of vaccinia, late promoters of polyoma, the lac the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage lambda, control regions of fd coat protein, 3-phosphoglycerate kinase promoter, acid phosphatase promoter, or promoters of yeast xcex1 mating factor.
Furthermore, the present invention extends to a unicellular host transformed or transfected with an expression vector of the present invention. Such a unicellular host can be selected from the group consisting of E. coli, Pseudonomas, Bacillus, Strepomyces, yeast, CHO, R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 and Sf9 cells.
Naturally, the present invention extends to a method of producing a mutant cation channel protein, comprising the steps of culturing a unicellular host transformed or transfected with an expression vector of the present invention under conditions that provide for expression of the isolated nucleic acid molecule of the expression vector and recovering the mutant cation channel protein from the unicellular host. Moreover, such a method can also be used wherein the expression vector comprises a an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated nucleic acid molecule comprising a DNA sequence of SEQ ID NO:17, or degenerative variants thereof, operatively associated with a promoter.
The present invention further extends to an antibody having a mutant cation channel protein of the present invention as an immunogen. More specifically, an antibody of the present invention can be a monoclonal antibody, a polyclonal antibody, or a chimeric antibody. Furthermore, an antibody of the present invention can be detectably labeled. Examples of detectable labels having applications in the present invention include, but are not limited to, an enzyme, a chemical which fluoresces, or a radioactive isotope.
Broadly, the present invention extends to a crystal of a cation channel protein having a central pore, which is found natively in a lipid bilayer membrane of an animal cell, such that the central pore communicates with extracellular matrix and cellular cytosol, wherein the crystal effectively diffracts x-rays to a resolution of greater than 3.2 angstroms.
Moreover, the present invention extends to a crystal of a cation channel protein as described above, wherein the cation channel protein comprises a first layer of aromatic amino acid residues positioned to extend into the lipid bilayer membrane proximate to the interface an extracellular matrix and lipid bilayer membrane, a second layer of aromatic amino acid residues positioned to extend into the lipid bilayer membrane proximate to the interface of cellular cytosol and said lipid bilayer membrane, a tetramer of four identical transmembrane subunits, and a central pore formed by the four identical transmembrane subunits.
Moreover, the present invention extends to a crystal of a cation channel protein described above, wherein each transmembrane subunit comprises an inner transmembrane alpha-helix which has a kink therein, an outer transmembrane alpha-helix, and a pore alpha-helix, wherein each subunit is inserted into the tetramer of the cation channel protein so that the outer transmembrane helix of each subunit contacts the first and second layers of aromatic amino acid residues described above, and abuts the lipid bilayer membrane. Moreover, the inner transmembrane helix of each subunit abuts the central pore of the cation channel protein, contacts the first and second layers of aromatic amino acid residues, is tilted by about 25xc2x0 with respect to the normal of the lipid bilayer membrane, and is packed against inner transmembrane alpha helices of other transmembrane subunits as the second layer of aromatic amino acid residues forming a bundle of helices at the second layer. The pore alpha-helix of each subunit is located at the first layer of said aromatic amino acid residues, and positioned between inner transmembrane alpha-helices of adjacent subunits, and are directed, in an amino to carboxyl sense, towards the center of the central pore.
Furthermore, the present invention extends to a crystal described above, comprising a cation channel protein having a central pore, which comprises a pore region located at the first layer of aromatic amino acid residues, and connected to the inner and outer transmembrane alpha-helices of said subunits. More particularly, the pore region comprises about 25-45 amino acid residues, a turret connected to the pore alpha-helix and the outer alpha-helix, wherein turret is located at the interface of said extracellular matrix and the lipid bilayer membrane. The pore region further comprises an ion selectivity filter connected to the pore alpha-helix and the inner transmembrane alpha-helix of each subunit. The ion selectivity filter extends into the central pore of the cation channel protein, and comprises a signature amino acid residue sequence having main chain atoms which create a stack of sequential oxygen atoms along the selectivity filter that extend into the central pore, and amino acid residues having side chains that interact with the pore helix. It is the signature sequence which enables a cation channel protein to discriminate among the cation intended to permeate the protein, and other cations, so that only the cation intended to permeate the channel protein is permitted to permeate.
The central pore further comprises a tunnel into the lipid bilayer membrane which communicates with the cellular cytosol, and a cavity located within the lipid bilayer membrane between the pore region and the tunnel, and connected to the them, such that the central pore crosses the membrane.
Furthermore, the structure of all ion channel proteins share common features, which are set forth in the crystal of a cation channel protein described above. Consequently, the present invention extends to a crystal of a cation channel protein having a central pore and structure, as described above, wherein the cation is selected from the group consisting of: Naxe2x88x92, K+, and Ca2+. Hence, the present invention extends to crystals of potassium channel proteins, sodium channel proteins, and calcium ion channels, to name only a few. In a preferred embodiment, the crystal of a cation channel protein comprises a crystal of a potassium ion channel protein.
In addition, a crystal of a cation channel protein of a present invention comprises the amino acid sequence of any presently known, or subsequently discovered cation protein channel. Consequently, the present invention extends to a crystal of a cation channel protein having a central pore, which is found natively in a lipid bilayer membrane of an animal cell, such that the central pore communicates with extracellular matrix and cellular cytosol, wherein the crystal comprises an amino acid sequence of:
residues 23 to 119 of SEQ ID NO:1 (Streptomyces lividans);
residues 61 to 119 of SEQ ID NO:2 (E. coli);
residues 61 to 119 of SEQ ID NO:3 (Clostridium acetrobutylicum);
residues 61 to 119 of SEQ ID NO:4 (Drosophila melanogaster);
residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:7 (Paramecium tetraaurelia);
residues 61 to 119 of SEQ ID NO:8 (C. elegans);
residues 61 to 119 of SEQ ID NO:9 (Mus musculus);
residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:11 (Arabidopsis thaliana);
residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or
residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);
or conserved variants thereof.
In a preferred embodiment, a crystal of the present invention having a central pore, which is found natively in a lipid bilayer membrane of an animal cell, such that the central pore communicates with extracellular matrix and cellular cytosol, comprises an amino sequence of amino acid residues 23 to 119 of SEQ ID NO:1, has a space grouping of C2, a unit cell of dimensions of a=128.8 xc3x85, b=68.9 xc3x85, c=112.0 xc3x85, and xcex2=124.6xc2x0.
Furthermore, the present invention extends to a crystal of a cation channel protein having a central pore, which is found natively in a lipid bilayer membrane of an animal cell, such that the central pore communicates with extracellular matrix and cellular cytosol, wherein the channel protein comprises a signature sequence comprising:
Thr-Val-Gly-Tyr-Gly-Asp xe2x80x83xe2x80x83(SEQ ID NO:15). 
In another embodiment, the present invention extends to a method for growing a crystal of a cation channel protein having a central pore, which is found natively in a lipid bilayer membrane of an animal cell, such that the central pore communicates with extracellular matrix and cellular cytosol, by sitting-drop vapor diffusion. Such a method of the present invention comprises the steps of providing the cation channel protein, removing a predetermined number of carboxy terminal amino acid residues from the cation channel protein to form a truncated cation channel protein, dissolving the truncated cation channel protein in a protein solubilizing solution, such that the concentration of dissolved truncated channel protein is about 5 to about 10 mg/ml, and mixing equal volumes of protein solubilizing solution with reservoir mixture at 20xc2x0 C. Preferably, the reservoir mixture comprises 200 mM CaCl2, 100 mM Hepes, 48% PEG 400, pH 7.5, and the protein solution comprises (150 mM KCl, 150 mM Tris, 2 mM DTT, pH 7.5).
Moreover, the present invention extends to a method of growing a crystal of a cation channel protein as described above, wherein a crystal can be grown comprising any kind of cation channel protein. In particular, the present invention can be used to grow crystals of potassium channel proteins, sodium channel proteins, or calcium channel proteins, to name only a few.
Furthermore, the present invention extends to a method of growing a crystal of a cation channel protein, as described herein, wherein the crystal comprises an amino acid sequence of:
residues 23 to 119 of SEQ ID NO:1 (Streptomyces lividans);
residues 61 to 119 of SEQ ID NO:2 (E. coli);
residues 61 to 119 of SEQ ID NO:3 (Clostridium acetrobutylicum);
residues 61 to 119 of SEQ ID NO:4 (Drosophila melanogaster);
residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:7 (Paramecium tetraaurelia);
residues 61 to 119 of SEQ ID NO:8 (C. elegans);
residues 61 to 119 of SEQ ID NO:9 (Mus musculus);
residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:11 (Arabidopsis thaliana);
residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);
residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or
residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);
or conserved variants thereof.
Numerous methods can be used to provide a cation channel protein, for use in growing a crystal. For example, traditional purification techniques such as gel filtration, HPLC, or immunoprecipitation can be used to purify cation channel proteins from the membranes of numerous cells. In another method, recombinant DNA technology can be used, wherein a nucleic acid molecule encoding the particular cation channel protein can be inserted into an expression vector, which is then used to transfer a unicellular host. After transfection, the host can be induced to express the nucleic acid molecule, and the particular cation channel protein can be harvested from the membrane of the unicellular host.
Moreover, numerous methods are available for removing a predetermined number of carboxy terminal amino acid residues from the cation channel protein to form a truncated cation channel protein. For example, chemical techniques can be used to cleave a peptide bond between two particular amino acid residues in the carboxy terminus of the cation channel protein. In another embodiment, the cation channel protein can be contacted with a proteolytic enzyme, so that the predetermined number of residues from the carboxy terminus are enzymatically removed from the carboxy terminus of the cation channel protein, forming a truncated cation channel protein. In a preferred embodiment, the cation channel protein comprises a potassium channel protein having an amino acid sequence of SEQ ID NO:1, which is contacted with chymotripsin so that residues 1-22 are removed, forming a truncated potassium channel protein comprising an amino acid sequence of residues 23-119 of SEQ ID NO:1.
This invention further provides for a prescreening method for identifying potential modulators of potassium ion channel function comprising the steps of: (i) binding a soluble potassium ion channel protein to a solid support where the ion channel has the scaffold of a two-transmembrane-domain-type potassium ion channel and has a tetrameric confirmation; (ii) contacting the soluble potassium ion channel protein in step i with a compound in an aqueous solution; and, (iii) determining the binding of the compound to the soluble potassium ion channel protein.
In addition, this invention provides for a method of screening for compounds which selectively bind to a potassium ion channel protein comprising: (i) complexing a functional two-transmembrane-domain-type potassium ion channel protein to a solid support; (ii) contacting the complexed protein/solid support with an aqueous solution said solution containing a compound that is being screened for the ability to selectively bind to the ion channel protein; and, (iii) determining whether the compound selectively binds to the ion channel protein with the provisoes that the potassium ion channel protein is in the form of a tetrameric protein; and, when the protein is mutated to correspond to the agitoxin2 docking site of a Shaker K+ channel protein by substituting amino acid residues permitting the mutated protein to bind agitoxin2, the protein will bind agitoxin 2 while bound to the solid support, said substituting of residues being within the 36 amino acid domain defined by xe2x88x9225 to +5 of the selectivity filter where the 0 residue is either the phenylalanine or the tyrosine of the filter""s signature sequence selected from the group consisting of glycine-phenylalanine-glycine or glycine-tyrosine-glycine.
In a particular embodiment of the method for screening for compounds as described above, a prokaryote two-transmembrane-domain-type ion channel protein is used, such as from Steptomyces lividans especially, the KcsA channel. The channels can be either wild-type or mutated from a wild-type protein. One mutation is confined to the 36 amino acid domain defined by xe2x88x9225 to +5 of the selectivity filter where the 0 residue is either the phenylalanine or the tyrosine of the filter""s signature sequence selected from the group consisting of glycine-phenylalanine-glycine or glycine-tyrosine-glycine. The method of this invention includes the use of channel mutations where the protein alteration involves the deletion of a subsequence of the native amino acid sequence and replacement of that native sequence with a subsequence from the corresponding domain of a second and different ion channel protein. The second ion channel protein can be from either a prokaryote or an eukaryote cell.
The methods described above may be conducted using an aqueous solution comprises a nonionic detergent.
In addition to the methods of this invention, the invention further comprises a column having the channel proteins of this invention bound thereto. The proteins are as described herein.
The invention also provides for a non-natural and functional two-transmembrane-domain-type potassium ion channel protein wherein the non-natural protein is mutated in its amino acid sequence from a corresponding natural protein whereby the mutation does not prevent the non-natural protein from binding agitoxin2 when the non-natural protein is further mutated to correspond to the agitoxin2 docking site of a Shaker K+ channel protein said docking site created by substituting amino acid residues selected from within the 36 amino acid domain defined by xe2x88x9225 to +5 of the Shaker K+ selectivity filter where the 0 residue is either the phenylalanine or the tyrosine of the filter""s signature sequence selected from the group consisting of glycine-phenylalanine-glycine or glycine-tyrosine-glycine. It is preferred that the non-natural protein so modified will binds to a channel blocking protein toxin with at least a 10 fold increase in affinity over the native ion channel. The non-natural proteins include those mutations described above for use on a solid support to identify modulators of potassium ion function.
The invention further provides for a means to assess the adequacy of the structural conformation of a two-transmembrane-domain-type potassium ion channel protein for high through put assays comprising the steps of: (i) complexing a two-transmembrane-domain-type potassium ion channel protein having a tetrameric form to a non-lipid solid support under aqueous conditions; (ii) contacting the complexed two-transmembrane-domain-type potassium ion channel protein with a substance known to bind to the two-transmembrane-domain-type potassium ion channel protein when bound to lipid membrane wherein the substance also modulates potassium ion flow in that channel protein; and, (iii) detecting the binding of the substance to the complexed two-transmembrane-domain-type potassium ion channel protein. The channel proteins can be wildtype proteins or modified as described above. Optionally the contacting is done in the presence of a non-ionic detergent and the substance for binding is either a channel blocker or other modulator including a toxin.
What""s more, the present invention extends to columns having applications in the methods of the invention. In particular, the present invention extends to a column comprising a solid support having bound thereto an ion channel having the scaffold of a two-transmembrane-domain-type potassium ion channel and having a tetrameric confirmation.
Furthermore, the present invention extends to a column as described above, wherein the ion channel is a non-natural and functional two-transmembrane-domain-type potassium ion channel protein wherein the non-natural protein is mutated in its amino acid sequence from a corresponding natural protein. Such a mutation does not prevent the non-natural protein from binding a toxin, such as agitoxin2 when the non-natural protein is further mutated to corresponding to the agitoxin2 docking site of a Shaker K+ channel protein. Numerous means are available to the skilled artisan to create the docking. A particular means to create the docking site comprises substituting amino acid residues selected from within the 36 amino acid domain defined by xe2x88x9225 to +5 of the Shaker K+ selectivity filter where the 0 residue is either the phenylalanine or the tyrosine of the filter""s signature sequence selected from the group consisting of glycine-phenylalanine-glycine or glycine-tyrosine-glycine.
Accordingly, it is a principal object of the present invention to provide a crystal comprising a cation channel protein.
It is another object of the present invention to provide a method for growing a crystal comprising a cation channel protein.
It is yet another object of the present invention to utilize information on the structure of a cation channel protein obtained from a crystal of the present invention, in an assay system for screening potential drugs or agents that may interact with a cation channel protein. Interaction of the potential drug or agent with a cation channel protein includes binding to a cation channel protein, or modulating the function of a cation channel protein, wherein modulation involves increasing the function of a cation channel protein to allow more specific cations to cross a cell membrane, or decrease the function of a cation channel protein to limit or prevent specific cations from permeating through the protein and crossing the cell membrane. Such drugs or therapeutic agents may have broad applications in treating a variety of abnormal conditions, such as cardiac arrhythmia, diabetes mellitus, seizure disorder, asthma or hypertension, to name only a few.
It is yet another object of the present invention to provide mutant form of a cation channel protein, preferably a potassium channel protein from Streptomyces lividans, which binds to Agitoxin2, a toxin found in scorpion venom, in a manner very similar to that in which eukaryotic potassium channel proteins bind to Agitoxin2. Consequently, a mutant cation channel protein of the present invention mimics a functional eukaryotic potassium channel protein, and can serve as a model therefor in screening potential drugs or agents that may interact with a eukaryotic potassium channel protein.
It is still yet another object of the present invention to provide a method of preparing functional cation channel proteins for use in screen systems for assaying potential drugs or therapeutic agents which may have applications in treating conditions related to the function of cation channel proteins in vivo.
It is yet another object of the present invention to provide mutated prokaryotic cation channel proteins which mimic eukaryotic cation channel proteins. With these mutated prokaryotic cation channel proteins, drugs or other can be screened for potential interaction with cation channel proteins in vivo, and hence, potential use as therapeutic agents in treating conditions related to the function of cation channel proteins in vivo, such as cardiac arrhythmia, diabetes millitus, seizure disorder, asthma or hypertension, to name only a few.